It is desirable for many purposes to increase (amplify) the amount of a nucleic acid sequence present in a sample. Detection and cloning of specific genomic or nucleic acid targets is limited by the ability to obtain sufficient starting material or by the abundance of the target sequence. Detection of a specific nucleic acid sequence is important in many fields and especially in clinical diagnostics, forensics, environmental and foodstuff monitoring and biological research.
It is known to prime reverse transcriptase in a random, non-specific manner for the synthesis of cDNA from mRNA (Noonan K. E. et al., Nucl. Acids Res. 16: 10366 (1988)). Noonan et al. disclose a method of mRNA phenotyping which combines reverse transcription of mRNA with polymerase chain reaction (PCR) amplification (infra) of the desired targets. The reverse transcriptase reaction was primed with random hexadeoxyribonucleotides rather than with the more commonly used oligo-dT primer so as to minimize the effects of sequence complexity, mRNA secondary structure and varying lengths of poly A tails. However, although reverse transcriptase was able to synthesize primary transcripts using primers of a random sequence, it was still necessary to amplify the specific cDNA product of interest with PCR for further analysis.
It is also known to prime DNA polymerase with oligonucleotides in a random non-specific manner for the synthesis of labelled or derivatized DNA probes for use in the detection of other nucleic acid sequences (Feinberg, A. P. et al., Anal. Biochem. 132:6-13 (1983); Liang, W. et al., Nucl. Acids Res. 16:3579 (1988)). According to this technique, DNA is first denatured by heating, so that the double stranded DNA becomes single stranded, and then random hexanucleotide primers are added, together with deoxynucleoside triphosphates, buffer, the Klenow fragment of E. coli DNA polymerase I, and a radioactive deoxynucleoside triphosphate, and incubated at room temperature for three to four hours. Although new DNA strands are synthesized which are complementary to the existing DNA template and which utilize the random oligonucleotides as primers, random primer DNA labelling by this technique does not substantially amplify the existing DNA. Calculations based on the data presented by Feinberg et al., supra, show that a maximum of a single copy of DNA synthesis occurs during the reaction period of many hours.
The PCR has become widely used as a nucleic acid amplification technique (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796 EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194). Although the PCR provides a method for achieving the amplification of a particular nucleic acid region in an unfractionated sample, the method has several disadvantages. First, PCR requires the identification and use of two different oligonucleotide probes, both of which must be highly specific for each sequence to be amplified. Sequence-specific oligonucleotide probes represent a considerable expense because they usually must be synthesized by chemical methods and because they are used in relatively large amounts in each reaction in order to maximize the efficiency of the amplification procedure.
Secondly, PCR is usually performed using sophisticated programmable equipment. The products of each extension reaction in PCR ordinarily are separated from their template strands by heat denaturation. The multiple (for example, 30-70) cycles of heating, rehybridization and primer extension may be manually performed, but more commonly, programmable temperature control devices are employed.
Thirdly, the PCR reaction is usually run in conjunction with a thermostable DNA polymerase. The combination of synthetic oligonucleotide primers, sophisticated equipment, and unusual DNA polymerase means that PCR, while powerful, is expensive.
Variations on the PCR technique have been reported which only partially address some of these problems. For example, Loh et al., Science 243:217-200 (1988), discloses a PCR technique which requires that the sequence of only one end of the target be known.
In many cases, sequence information about a target is not known although a clone to that target is available. Also, in some cases, the sequence may be highly variable, so that it is difficult if not impossible to identify target-specific oligonucleotide probes.
Thus, a need exists for a method capable of amplifying the levels of a nucleic acid sequence wherein such method does not depend on the availability of sequence information or the identification of target-specific oligonucleotides. Further, it is desirable that such a method would not require complex sample processing equipment or technical manipulation during the amplification. It is also desirable that the method have low background levels, i.e. it should polymerize little or no nucleotides in the absence of a polynucleotide target.